Role of Afadin Signaling in Nephron Tubulogenesis Project Summary/Abstract Formation of polarized epithelial tubules is central to the structure and function of the kidney. Despite its importance, there is a fundamental gap in understanding the earliest steps in tubulogenesis, namely how epithelia establish apical-basal polarity and generate a central lumen. Continued existence of this gap represents an important problem because, until it is filled, an understanding of developmental abnormalities of the kidney will remain largely incomprehensible. The objective of this application is to elucidate how apical- basal polarity is established and lumen formation initiated, focusing on the role of Afadin signaling in nephron formation. Afadin is an adaptor protein to the Nectin family of adhesion receptors. Our preliminary data identify Afadin as critical for establishing an apical surface and initiating lumen formation in developing mouse nephrons. To further elucidate the cellular and molecular mechanisms that initiate polarity and lumen formation, the proposed studies have three specific aims. The first aim is to characterize molecular steps of lumen formation in vivo and in great detail. These studies will generate a molecular timeline of lumen initiation through subcellular localization of polarity, trafficking, and junctional complexes, and through live imaging of lumen formation in mice. Once characterized, additional studies will begin to assign proteins to different stages in the pathway using mouse models. The second aim is to identify novel molecular mechanisms of apical polarity and lumen formation using an established 3D cell culture model. Specifically the proposed experiments will elucidate a detailed signaling pathway by which Nectins and Afadin initiate lumen formation. The third aim will examine the role of Afadin in tubule regeneration after ischemic injury, emphasizing similarities to the developmental process. Together these studies will delineate fundamental steps in polarity and lumen initiation and provide an improved framework for understanding developmental and acquired disorders of renal tubules.